Human APOBEC3G (hA3G) is a potent antiviral host defense protein that functions in post-entry HIV restriction via cytidine deaminase-dependent and -independent mechanisms. The HIV viral infectivity factor (Vif) overcomes hA3G activity by binding to it and mediating its destruction via polyubiquitination and proteosomal degradation. Recent small angle X-ray scattering (SAXS) and biochemical studies from the applicant's lab enabled an empirical determination of the hA3G molecular shape in solution, which revealed an elongated catalytically active, tail-to-tail dimer. These observations enabled the identification of folding domains of hA3G that could be expressed as soluble proteins and evaluated for their contribution to structure and activity. The SAXS analysis also suggested that dimers of hA3G could be bridged by short RNA strands to yield catalytically inactive oligomers, which is a consequence of hA3G's innate nucleic acid binding ability. The central hypothesis of this proposal is that (i) dimeric and higher order forms of hA3G represent `core' structures whose relative abundance determines whether cells are permissive to HIV infection, and (ii) the HIV protein Vif must interact with hA3G in the context of these structures to inactivate this host defense factor. As such, the determination of hA3G7Vif complexes at high resolution will advance our ability to disrupt the host/virus protein interface through knowledge-based drug design. The Aims for the R21 phase are: (1a) To prepare the hA3G7Vif complex & domains thereof to define crystallization conditions; (1b) To evaluate intact hA3G binding to defined nucleic acids to obtain soluble, monodisperse material for biophysical studies, and (2) To determine the molecular envelope of Vif and/or hA3G7Vif complexes by SAXS in order to complement crystallographic studies. Milestones for evaluating progression into the R33 phase are: (i) attainment of crystals of hA3G, Vif, or domains thereof or an hA3G7Vif complex, and (ii) determination of SAXS envelopes of hA3G7Vif complexes. In the R33 phase we will leverage knowledge gained from the R21 phase to solve crystal structures. The Specific Aims of the R33 phase are: (3) To determine structures of individual components or complexes from Aim 1. If hA3G7Vif crystal structures are not available, the domains of individual host or viral crystal structures will be docked/fitted together computationally using the hA3G7Vif SAXS envelope from Aim 2; and (4) To use structural information to identify key amino acids that stabilize the host/virus molecular interface, and to validate these observations by biochemical and functional endpoint assays. Collectively the results will identify key amino acids necessary for Vif binding to hA3G. This information will provide a platform for rational drug design that will lead to a novel class of HIV/AIDS therapeutics to improve public health.Public Health Relevance Statement The human protein APOBEC3G is a natural, anti-HIV defense factor that becomes inactivated through association with the HIV protein Vif. The goal of this proposal is to visualize how Vif recognizes APOBEC3G at the atomic level. By observing the chemical attributes of the host/virus protein-protein interface, researchers will be able to create novel HIV/AIDS therapeutics that shield APOBEC3G from Vif, thereby promoting the body's own anti-HIV defense system.